Systematic Genetic Analysis of Fungal Virulence

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DESCRIPTION (provided by the applicant): We seek to harness complete genome sequences to understand host-pathogen interactions that determine the ability of a fungal pathogen to cause disease in a mammalian host. The opportunistic pathogen Cryptococcus neoformans is a major cause of death in individuals with T lymphocyte defects, and is estimated to be the proximal cause of mortality in 5-10% of the 3 million deaths; caused by AIDS annually. C. neoformans is an excellent model organism for understanding fungal virulence because of its molecular manipulability, haploid genetics and faithful animal infection models. We have annotated the C. neoformans genome serotype A genome sequence, generated signature-tagged knockout mutants by gene targeting for -1200 genes (corresponding to about 20% of the genome), constructed and tested a whole-genome oligonucleotide DMA microarray, and initiated genetic screens for virulence factors in vitro and in a mouse in vivo infection model. We seek to continue to develop and utilize these tools to develop a comprehensive picture of pathogen genes necessary for virulence and to identify and to initiate more detailed studies of the subset involved in invasion of the central nervous system by C. neoformans. To achieve these goals we 1) complete construction of the C. neoformans knockout collection, 2) perform in vitro characterization of the knockout collection, 3) screen knockout collection for virulence mutants using a mouse model, and 4) identify and characterize mutants defective in invasion of the central nervous system. By systematically identifying molecules produced by C. neoformans important for virulence and by understanding how they function, we hope to lay the groundwork for the discovery of new therapies to treat fungal infections.